1. Field of the Invention
The present invention is related to a centrifugal separator and a centrifugal rotor and also is related to a centrifuge separation method suitable for separating a sample solute from a solvent and recovering them individually using the centrifugal separator.
2. Description of the Related Prior Arts
Various efforts are being made to elucidate the mechanism of life by measuring polynucleotides such as DNAs and mRNAs in living organisms. Main practical approaches supporting these efforts include; (1) prove testing, which involves preparing a DNA prove with its sequence complementary with a target DNA one another, for determining whether the DNA prove can be hybridized with the target DNA and (2) PCR testing, which requires selection of a region where the target DNA sequence runs, for determining whether a fragment can be PCR-amplified using a pair of DNA primers or for measuring the length of the sequence of the amplified segment and examining the sequence itself.
Since the mechanism of living organs works by means of expression of a wide variety of genes while associating with each other, transcription of the genes contained in chromosomes must be comprehensively examined. In making an attempt to elucidate the mechanism of the development of cancer and hereditary diseases from an aspect of DNAs, it is required to compare mRNAs or identify extensively any difference(s) between normal and variant cells. To measure the mRNAs, first, the mRNAs are extracted from the cells, cDNAs are prepared using reverse transcriptase, and then the specific mRNAs are detected using prove testing or PCR. The methods getting a lot of attraction include Differential Display (Peng Liang and Arthur B. Pardee, Science 258, 967-972 (1992)), by which the mRNAs are compared between the cells or tissues, and Amplified Fragment Length Polymorphisms (WO093/06239). Based on PCR, the latter method amplifies the mRNAs using random primers or arbitrary sequence primers to get their patterns and compares the resulting patterns, allowing the transcription of the mRNAs between the cells or the tissues to be matched.
On the other hand, the Finger Printing method focusing on genome or a specific DNA region has been used on a pilot basis. The Restriction-Enzyme Landmark-Scanning method, first, uses NotI, a rare cutter, to cut the genome into cutting sites, in which labels are introduced. Second, the resulting cutting sites with labels are separated by agarose electrophoresis. The DNAs separated by agarose electrophoresis are further cut into smaller fragments in a gel using a 4-base restriction recognition enzyme and then an agarose gel is spread on a polyacrylamide slab gel. This means that this method is designed to detect a wide variety of genome-derived DNA fragments by 2-dimensional electrophoresis.
In the fields around genome analysis, a need has been increasingly boosted for a method of higher efficient determination of DNA sequences. In place of conventional, manual-based methods of determination of DNA sequences, by which the DNA fragments are labeled using a radioisotope and the lengths of the DNAs are measured by gel electrophoresis, a device (a DNA sequencer), which labels the DNAs with a fluorophore to optically auto-detect the DNA fragments by irradiating a light beam while gel electrophoresis is proceeding, and the determination method of DNA sequences with the DNA sequencer have being spread. The determination methods of DNA sequences, called the Sanger and Dideoxy Chain-Termination methods, are those by which a DNA oligomer, a primer, is hybridized with a target DNA, various lengths of DNA fragments are prepared to be used in determination of DNA sequences by complementary-strand synthesis using an enzyme, and the lengths of the DNA fragments are measured by gel electrophoresis to determine the DNA sequences. The total base length to be determined at the same time by the determination method of DNA sequences may span over from 400 to 700 bases depending on the ability of the gel to separate the fragments. Note that since the determination methods target largely at the genomes and mRNAs and in many cases, the base length to be determined is several kilobases for the mRNAs and is longer than several kilobases for the genomes, respectively, the sequencer cannot determine the entire DNA sequence at the same time.
Conventionally, the Shotgun method has been used for determination of long DNA sequences with several kilobases to several tens kilobases. In case of the Shotgun method, the DNAs are randomly cut into fragments using, for example, an ultrasonic wave, the DNA fragments are cloned and embedded into culture media such as Escherichia coli, and then after colonial cultivation, Escherichia coli is cultivated in each colony to increase the number of DNA fragment copies. Subsequently, sample DNAs are extracted and DNA analysis, for example, determination of DNA sequences, is carried out. In principle, the Shotgun method, which randomly controls the DNA fragments to remove any overlapping between the DNA fragments, leading to elucidation of a linkage between the DNS fragments, is suitable for the long DNA strands with their sequences unknown and used in the Genome Sequencing Project as a primary method.
The examinations using a DNA prove or PCR, Differential Display, analysis of amplified fragment length polymorphisms, Restriction-Enzyme Landmark-Scanning method, and the determination method of DNA sequences mentioned above are implemented be means of auto-measurement instrumentation with electrophoresis and fluorescent detection by laser irradiation combined therein or auto-hybridization detection instrumentation.
On the other hand, since sample preparation exploiting skills of molecular biology for gene analysis and gene diagnosis involves various processes such as purification of nucleic acid and hydrogen reaction, it is often required that liquid samples should be handled on a minute scale of micro litter. Liquid sample handling necessary for preparation of these samples comprises quantification, transport, retention, mixture, and storage, for each of which a suitable liquid-sample handling tool is commercially available.
As a handling tool for quantification and transport of the liquid samples, a micropipette with a plastic chip is widely used. The micropipette can be used to suck and discharge the liquid sample through an air cylinder using a disposal, plastic chip tube. Plastic sample tubes and multi-well plates are commonly used for retention, mixture, and storage of liquid. Intended to handle liquid samples during purification, in particular, column vessels with a filter are widely spread. Full-automatic equipment, which prepares the samples using these devices and jigs, has been introduced in the market.
Conventionally, the ethanol precipitation method has been usually used for manual preparation of the samples. In case of the ethanol preparation method, the DNAs or the RNAs are precipitated and separated by adding ethanol to prepare a 60-70% of DNA or RNA sample solution in a certain ion environment, followed by centrifugation. Alternately, to remove proteins and lipids from living samples for purification of DNAs or RNAs, the technique, by which phenol is added to a sample mixture to denature and precipitate proteins or by which the lipids are removed by the chloroform extraction method, has been usually used. These techniques requiring centrifugation are essential to molecular biology.
In relation to conventional centrifugation, first of all, centrifugal tubes having samples loaded are mounted on the centrifugal rotors of the centrifugal separator. A batch processing, in which multiple centrifugal tubes are processed at the same time, is essential to centrifugation. In many cases, the quantity of a sample liquid depends on the kind of the sample and when the multiple samples are centrifuged at the same time, the centrifugal tubes associated with the individual samples must have been balanced (the weights of two centrifugal tubes are manually adjusted to be equal to each other) or the centrifugal separator with a auto-balance mechanism must have been used. Furthermore, for the multiple centrifugal tubes having samples loaded to be automatically mounted on the centrifugal rotors of the centrifugal separator one by one, the following steps must be taken; 1) the centrifugal tubes are positioned on the centrifugal rotors, 2) the centrifugal tubes are picked up, 3) a given number of centrifugal tubes are mounted on the centrifugal rotors one by one, and then 4) the centrifugal rotors are started, 5) the rotating centrifugal rotors are stopped and aligned with heir associated stop positions, and finally, 6) the multiple centrifugal tubes are taken out from the centrifugal rotors one by one in the order of mounting on.
Recently, a flow-through type micro-centrifuge, in which the centrifugal tubes can be arrayed for high-throughput instrumentation, has been reported (A. Marziali et al. xe2x80x9cAn arrayable flow-through microcentrifuge for high-throughput instrumentationxe2x80x9d (Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 61-66 (1999))). The report is summarized as follows; with multiple high-speed rotors also serving as sample holders introduced, a miniature flow-through type micro-centrifuge, which achieves high-throughput centrifugation of a large number of samples, has been developed. The small-size rotors of the flow-through type micro-centrifuge are arrayable on a standard microtiter plate having a 96-well spacing, which makes them suitable for an automated processor capable of parallel processing of the multiple samples. The flow-through type micro-centrifuge could be used in place of a standard type centrifuge in various processes, in which only a small number of samples are processed. Techniques have been developed for recovering both supernatants and pellets, as well as for sample mixing and cleaning of the reusable rotors. The report further discusses the schema of applications of the flow-through type micro-centrifuge not only to cell separation and re-suspension but also to DNA purification and condensation, and its implementation method.
The flow-through type micro-centrifuge developed by A. Marziali et al. allows the rotors to rotate at a higher speed, each of which has a space with a V-shape cross section, an upper hole passing through to the V-shape space at its upper part, and a lower hole passing through to the V-shape space at its lower part. When sample solutions containing the samples are injected from the upper holes while the rotors are rotating, both the samples and solvents are forced to move toward the sidewalls of the V-shape spaces by a centrifugal force. When the rotors are stopped, the solvents flow out from the lower holes, while the samples are captured at the sidewalls of the V-shaped spaces.
Since not only the samples centrifuged through the centrifugal process are sensitive to any shock but also centrifuged precipitates are easy to re-mixed with the solvents or to float, a special attention should be paid in handling the centrifuged samples. To automate the centrifugal process, advanced sensing and handling techniques are required, while practical problems with cost and accuracy remain unresolved, preventing a full automatic device, which performs a course of sample preparation processes with the centrifugal process combined automatically, from being building. Furthermore, in considering a full-automatic device integrating an automatic sample preparation process or a measuring system, a problem of batch processing brings out as a deterministic bottleneck. The processes other than centrifugation in sample preparation can be sequentially treated (discretely treated), which means that all the individual samples can be separately processed. An advantage of discrete treatment lies in that the process can be advanced on an assembly-line basis, that it is suitable for automation of the process, and that any interrupt processing is allowed. On the contrary, in case of batch processing, a given batch process has been complete before the next process can start. This imposes such a limitation on batch processing that samples should be supplied from the upper holes, which does not meet a requirement by researchers for rapid availability.
Likewise, since the flow-through type centrifugal separator developed by A. Marziali et al., which is equipped with a different rotor for each of individual samples, has the same array as that of a 96-well micro-plate, it can perform batch processing only. Furthermore, since the flow-through type centrifugal separator has an inlet in the upper part of each rotor, from which a sample is injected, an outlet in the lower part of each rotor, from which the sample is recovered, and a tubing structure connecting between the inlet and the outlet, sample solutions must be injected from the upper inlets in the upper parts of the rotors while the rotors are rotating.
In the biochemical field, considering automation of sample preparation, the system capable of discretely treating all the processes is more preferable than the batch-processing system. No meaningful data could be obtained simply by determining DNA sequences of individual samples as in conventional sample pre-treatment used for determination of DNA sequences in the Human Genome Project and it was required to obtain a certain range of genomes and mRNA sequences together, batch processing capable of parallel-processing multiple samples at the same time was essential to sample preparation. On the other hand, for example, as the Genome Project advances, it will become important to compare narrow regions of the corresponding genomes between heterogeneous solid matters or biological species. Fundamentally, data for each sample will be more significant and probably a requirement will be augmented for rapid sample preparation satisfying needs for urgent assay. Since it is also expected that the number of samples will increase, a method, which successively gives samples processed at a given time interval, is preferable for the system integrating measurement and data processing, which provides higher flexibility and makes system integration easy.
The discrete treatment process, in which usually, sample vessels are successively fed into individual processes of sample preparation one by one, allows easy insertion of sample vessels into a specified different process and has almost no effect on the entire system, that is on the entire processing of all the samples except for a time delay of one process with respect to the samples to be inserted.
An objective of the invention is to provide a centrifugal separation method, centrifugal rotors, and a centrifugal separator, which cooperatively allow discrete treatment (sequential treatment) and to provide, in particular, the centrifugal separation method and the centrifugal separator, which enable discrete-treatment for recovering and purifying the precipitants of biogenic samples by adding an organic solvent, especially for recovering DNAs and RNAs. Furthermore, another objective of the invention is to provide a sample preparation device suitable for molecular biological samples using a centrifugal separator, which enables discrete treatment, and a sample preparation method using it.
In the centrifugal separator of the invention, sample solutions are added into separation chambers, each of which is disposed in every centrifugal rotor, while the centrifugal rotors remain stationary, upper openings of the centrifugal rotors are closed and then the centrifugal rotors are rotated for centrifugation, and finally individual samples are discretely treated in their associated centrifugal rotors. With respect to the centrifugal rotors having a structure suitable for discrete treatment, the centrifugal separator allowing discrete treatment using the centrifugal rotors, and the discrete treatment sequence, the configuration of the invention is characterized in that:
(A) The centrifugal rotor of the invention has a configuration, in which the upper opening is formed along an axis of symmetry, a rotation axis of the centrifugal rotor (hereafter, simply referred to as the first direction (axis Z), only one sample separation chamber passing through to said upper opening being disposed in the centrifugal rotor. According to the invention, one centrifugal rotor centrifugally separates a given sample therein independently of other samples.
The sample separation chamber and the centrifugal rotor have two symmetric planes, respectively, which intersect with each other, including the rotation axis of the centrifugal rotor.
Assuming that two directions intersecting with the first direction are the second direction (axis X) and the third direction (axis Y), respectively, the precipitants are deposited at the ends of the sample separation chamber in the third direction through centrifugation by making longer the length of the sample separation chamber in the third direction than that in the second direction.
This means that it is assumed that the direction, in which the distance between the ends of the sample separation chamber in the direction normal to the rotation axis of the centrifugal rotor is the maximum, is axis Y (the third direction) and the direction intersecting with axes X and Z is axis X (the second direction).
To facilitate production of the precipitants such as DNAs by centrifugation, the structure of the sample separation chamber, into which a sample to be centrifugally separated is injected, is adjusted so that with respect to the cross section of the sample separation chamber parallel to the XY plane, the cross section at a distance far from axis Z is smaller than that at a distance near axis Z. Furthermore, the sample separation chamber has a concave portion at the bottom with its symmetric planes intersecting with each other including the rotation axis of the centrifugal rotor. When the centrifugal rotor is stopped after centrifugation has been finished, a supernatant liquid obtained by centrifugation is deposited in the concave portion. The supernatant liquid is sucked and discharged from the upper opening of the sample separation chamber.
Subsequently, when a cleaning liquid is added in the sample separation chamber from the upper opening for cleaning the precipitants and the centrifugal rotor is started, the cleaning liquid gets into touch with the precipitants and the precipitants are cleaned. The centrifugal rotor is stopped and the cleaning liquid is sucked out and discharged from the upper opening. Likewise, when a dissolving liquid is added in the sample separation chamber from the upper opening for dissolving the precipitants and the centrifugal rotor is started, the dissolving liquid gets into contact with the precipitants and the precipitants are dissolved. The centrifugal rotor is stopped and the solution containing dissolved precipitants thereof, which is a final product, is sucked out and recovered from the upper opening.
Thus, this simple configuration, in which the sample separation chamber is disposed in the centrifugal rotor, enables easy and speedy cleaning, re-dissolution, and recovery.
(B) The centrifugal rotor of the invention has a configuration, in which the upper and lower openings, both of which run along the symmetry axis, the rotation axis of the centrifugal rotor (in the first direction (axis Z)), only one sample separation chamber passing through to the upper and lower openings is disposed in the centrifugal rotor, and a solution holding vessel having the concave portion for retaining the sample solution to be centrifuged is laid out at the center of the inside of the sample separation chamber. Like that in (A), according to the invention, one centrifugal rotor centrifugally separates a given sample therein independently of other samples.
The solution holding vessel and the sample separation chamber have two symmetric planes, respectively, which intersect with each other, including the rotation axis of the centrifugal rotor. The solution holding vessel is a plate-like concave portion disposed fixedly in the centrifugal rotor. Assuming that two directions intersecting with the first direction are the second direction (axis X) and the third direction (axis Y), respectively, the precipitants are deposited at the ends of the sample separation chamber in the third direction through centrifugation by making longer the length of the sample separation chamber in the third direction than that in the second direction.
This means that it is assumed that the direction, in which the distance between the ends of the sample separation chamber in the direction normal to the rotation axis of the centrifugal rotor is the maximum, is axis Y (the third direction) and the direction intersecting with axes X and Z is axis X (the second direction). A pair of ends of the solution holding vessel are combined with an internal wall in the second direction of the sample separation chamber, while another pair of ends of the solution holding vessel are separated from an internal wall in the third direction of the sample separation chamber without getting into contact with each other. To facilitate production of the precipitants such as DNAs by centrifugation, the structure of the sample separation chamber, into which a sample to be centrifugally separated is injected, is adjusted so that with respect to the cross section of said sample separation chamber parallel to the XY plane, the cross section at a distance far from axis Z is smaller than that at a distance near axis Z. When the centrifugal rotor is stopped after centrifugation has been finished, a supernatant liquid obtained by centrifugation is discharged from the lower opening. According to the second embodiment of the invention, a sample can be added in the solution holding vessel from the upper opening and the supernatant liquid obtained by centrifugation is recovered into a waste vessel from the lower opening.
This means that when the centrifugal rotor is stopped, the sample can be injected into the solution holding vessel for retention, while when the centrifugal rotor is started, the sample solution moves into the sample separation vessel from the solution holding vessel in the radial direction along the rotation axis of the centrifugal rotor, the precipitates are deposited at the ends of the sample separation chamber in the third direction through centrifugation and retained there. When the centrifugal rotor stops, a supernatant liquid produced by centrifugation is discharged from the lower opening into a waste vessel. Subsequently, when a cleaning liquid is added in the solution holding vessel from the upper openings for cleaning the precipitants and centrifuged, the cleaning liquid moves into the sample separation chamber and gets into touch with the precipitants and the precipitants are cleaned. When the centrifugal rotor is stopped, the cleaning liquid is automatically discharged from the lower opening. Likewise, when a dissolving liquid is added in the solution holding vessel from the upper opening for dissolving the precipitants and centrifuged, the dissolving liquid moves into the solution holding vessel and gets into contact with the precipitants and the precipitants are dissolved. When the centrifugal rotor is stopped, the solution containing dissolved precipitants thereof, which is a final product, is automatically charged and recovered from the lower opening.
Thus, this simple configuration, in which the sample separation chamber is disposed in the centrifugal rotor, enables easy and speedy cleaning, re-dissolution, and recovery.
(C) According to the centrifugal separator of the invention, as described in the configurations (A) and (B), the centrifugal rotor is rotated by revolving a cover having a tip, which can be closely engaged with the upper opening for coupling. This means that the centrifugal separator has a configuration, in which engagement of the cover having a motor attached with the upper opening for coupling enabling a rotation moment of the motor to be transmitted to the centrifugal rotor for driving is used as the means for rotation driving. The centrifugal rotor is held by bearings disposed on a periphery around a bottom of the centrifugal rotor, allowing discharge of the waste liquid or the sample solution from the lower opening. According to the invention, a power for driving the rotation system of the centrifugal rotor is supplied from upper part instead of from the bottom as in the conventional concept.
The centrifugal separator suitable for automation can be implemented by combining the configurations (B) and (C), which prevents the samples to contaminate each other, allows the samples to be injected from the upper opening, and after centrifugation has been finished, the sample, a final product, can be recovered. In the time except for those when a sample is added, when a centrifugally-separated supernatant liquid is discharged, when the target precipitants are cleaned, re-dissolved, and recovered, every centrifugal rotor has the cover fitted in its upper opening, preventing the samples from contaminating each other, which often happens to become a problem.
(D) The centrifugal rotor according to the invention, as described in any of the configurations (A), (B), and (C), has a configuration, in which the centrifugal rotor and the sample separation chamber have two symmetric planes, respectively including the rotation axis of the centrifugal rotor, the centrifugal rotor comprises upper and lower parts, and the solution holding vessel is held fixedly in the sample separation chamber of the centrifugal rotor.
Like the configurations (A), (B), and (C), the centrifugal rotor described here has a configuration, in which engagement of the cover having a motor attached with the upper opening formed in the upper part for coupling enabling the rotation moment of the motor to be transmitted to the centrifugal rotor for driving is used as the means for rotation driving. Alternately, according to the configuration (A) of the invention, if the centrifugal rotor comprises both the upper and lower parts, the concave portion is formed having a symmetric axis corresponding to the rotation axis of the centrifugal rotor at the bottom of the lower part. The concave part does not penetrate into the sample separation chamber. It may have the configuration, in which engagement of the cover having a motor attached with the upper opening formed in the upper part for coupling enabling the rotation moment of the motor to be transmitted to the centrifugal rotor for driving is used as the means for rotation driving. Alternately, it has the configuration, in which the direct connection of the motor to the member formed at the bottom of the lower part to transmit the rotation moment of the motor to the centrifugal rotor is used as the means of rotation driving for rotating the centrifugal rotor. Thus, in the configuration (A), the centrifugal rotor can be rotated from any of the upper and lower parts.
(E) The sample preparation device has multiple centrifugal rotors as described in (A)-(D), for each of which the rotation driving system is separately controlled and sample addition, centrifugation, and sample recovery and the like are performed individually. Each of the centrifugal rotors is held in single transport device moving on a given trajectory and can move between a sample addition device and a sample recovery device, enabling sample addition, centrifugation, and sample recovery to be performed for each of centrifugal rotors individually. A mechanism for moving the transport device holding the centrifugal rotor along a guide in a given direction is provided and at a defined interval of the guide, each of the centrifugal rotors is rotated for centrifuging the sample, giving the ability of centrifuging the samples at a desired yield for a given time period. The guide may be formed into a loop shape such as a circle or ellipsoid, each of the centrifugal rotors is moved on the closed loop of trajectory in the given direction, and rotates in the given range of the predetermined, closed loop of trajectory for centrifuging the sample. In the vicinity of the guide, the sample addition devices, the sample recovery devices, and the centrifugal rotors are disposed and the individual centrifugal rotors rotate at the intervals between the sample addition devices and the sample recovery devices for centrifuging the samples. This practically allows an unlimited number of samples to be centrifuged.
(F) According to the sample preparation method of the invention, the discrete treatment sequence can be followed, in which the centrifugal rotors and the multiple rotation driving means for driving the rotation systems of the centrifugal rotors, as described (A)-(D), are used, respectively, the individual centrifugal rotors are moved along the guide in the given direction, and the rotation driving system of the individual centrifugal rotors are independently controlled, enabling separate sample addition, centrifugation, and sample recovery for each of the centrifugal rotors. In the vicinity of the guide, the sample addition device and the sample recovery device are disposed and the individual centrifugal rotors rotate at the intervals between the sample addition devices and the sample recovery devices for centrifuging the samples. In the discrete treatment sequence of the invention, a process, in which the samples are injected into the centrifugal rotors using the sample addition devices, a process, in which the centrifugal rotor is moved along the guide on the trajectory loop for centrifuging the sample, and a process, in which the supernatant liquid obtained by centrifugation is discharged using the solution discharging device, are sequentially performed for each centrifugal rotor. Furthermore, not only the sample addition device and the solution discharging device are disposed on the guide but also a solvent addition device is placed in the vicinity of the guide, which enables the process, in which the sample is injected into the centrifugal rotor, the process, in which the centrifugal rotor is moved along the guide on the trajectory loop to produce the precipitants by centrifugation, the process, in which the supernatant liquid obtained by centrifugation is discharged using the solution discharging device, the process, in which a solvent is added using the solvent addition device, the precipitates obtained by centrifugation are dissolved into a solute, and the process, in which the solute containing dissolved precipitants is recovered in the sample recovery device to be independently performed for each of the centrifugal rotors.
According to the invention, since one kind of sample is treated in single rotor, a problem of vexatious complication with the rotors of the centrifugal separators manufactured by the conventional arts, for example, positioning of multiple centrifugal tubes is eliminated and automatic sample preparation including centrifugation is facilitated. Furthermore, according to the invention, an unlimited number of samples can be practically centrifuged.